Terminalia catappa aqueous leaf extract reverses insulin resistance, improves glucose transport and activates PI3K/AKT signalling in high fat/streptozotocin-induced diabetic rats

Rising prevalence of type 2 diabetes mellitus (T2DM) in sub-Saharan Africa has necessitated surveys of antidiabetic medicinal plants. This study assessed the antidiabetic mechanism of Terminalia catappa aqueous leaf extract (TCA) in high fat/low dose streptozotocin-induced type 2 diabetic rats. T2DM was induced by a combination of high-fat diet and low dose STZ (30 mg/kg bw) and the animals were administered with TCA (400 and 800 mg/kg bw) orally daily for 28 days. Biochemical parameters and indices for diabetes including renal function tests and pancreatic histology were evaluated. Relative expression of hepatic insulin resistance, signalling and glucose transport genes were also assessed. Induction of T2DM resulted in significant (p < 0.05) weight loss, dysregulated glucose level and clearance, electrolyte imbalance and disrupted diabetic biochemical parameters. Diabetes onset also perturbed β-cell function and insulin resistance indices, damaged pancreas microanatomy, while disrupting the expression of insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and glucose transporter isoform 4 (GLUT-4) mRNA. Oral treatment of diabetic animals with TCA significantly (p < 0.05) ameliorated alterations due to T2DM induction in a manner comparable with glibenclamide. These results suggest TCA exerts its antidiabetic action by reversing insulin resistance, improving glucose transport and activating PI3K/AKT signalling.

Diabetes mellitus (DM) is a metabolic disease with characteristic high plasma glucose concentration due to either insulin deficiency and/or impaired insulin activity. Insulin release and action are affected by numerous factors such as lifestyle activities, genetics and epigenetics predisposition 1 . About 6.7 million and 416,000 deaths worldwide and Africa respectively, were associated with DM, ranking it among 90th percentile of diseases that cause death in year 2021 2 . Type 2 diabetes mellitus (T2DM) is the most predominant type of DM, which has constituted a public health challenge by contributing to poor quality of life, morbidity and mortality 3 . The prevalence and incidence of T2DM in Nigeria and sub-Sahara Africa are increasing at an alarming rate requiring an immediate solution 4,5 . Weight loss, polyuria, polydipsia, and hyperglycaemia are the common symptoms associated with T2DM. The β-cells of the pancreas are most affected during the onset of the disease, leading to dysregulated blood glucose levels and uptake with aberrant insulin secretion and action 6 .
Signal transduction of insulin (IST) is a mechanistic pathway involving mediators and enzymes that facilitate glucose entry into tissues and organs via glucose transporter type 4 (GLUT-4) 7,8 . The binding of insulin to the α chain of its receptors (IRs) initiates IST by inducing structural changes in the β chain via autophosphorylation at three tyrosine residues 9 . Hence activating its autokinase activity, leading to the phosphorylation and recruitment of different adaptor proteins (insulin receptor substrates (IRS), Shc protein and adapter protein (APS) Plant specimen collection, identification and preparation. T. catappa (TC) mature leaves were plucked from Covenant University, Ota, Nigeria. They were authenticated by Dr. J. O. Popoola and deposited at the Forestry Research Institute of Nigeria (FRIN) (FHI 112775). Crude TC aqueous leaf extract (TCA) was prepared using a rotary evaporator (Stuart RE 300/MS, Staffordshire, UK) to concentrate the aqueous filtrate as described by Iheagwam et al. 25 . The leaves were dried under shade in a well aerated room for two weeks, pulverised into a fine blend and steeped in distilled water (5% w/v) for 3 days before the aqueous filtrate was obtained. Studies complied with local and national ethical guidelines/regulations on the usage of plants.
Experimental animals and treatment dosage determination. Thirty Wistar rats of male sex (200 ± 20 g) aged between 6 and 8 weeks were purchased from the College of Medicine, University of Lagos for this study and acclimatised for two weeks prior to the experiment. Optimal living conditions were maintained, such as a humidity of 50 ± 5%, room temperature of 23 ± 2 °C and 12 h light/dark cycle in addition to food and water provision ad libitum. Covenant University Health, Research and Ethics Committee (CHREC/031/2018) approved the experimental protocol following institution guidelines for animal care and handling documented by ARRIVE guidelines and the National Institutes of Health (NIH). A cup of T. catappa juice measuring about 400 mL for an adult was suggested by herbal practitioners and ethnobotnical survey first thing in the morning and last thing at night for effective management of hyperglycaemia and diabetes 23 . A weight of 4.42 g of TCA was obtained after rotary concentration of the juice (400 mL). The dosage was then extrapolated to give 391.5 mg/ kg bw PO for albino rats.
Induction of diabetes and research design. The method previously described by Iheagwam,et al. 21 was used to induce T2DM using a combination of a high-fat diet (HFD) and low dose STZ (30 mg/kg bw). Experimental animals (n = 30) were randomly placed in five (5) groups containing 6 rats each, as earlier reported in a previous study by Iheagwam et al. 21 as depicted in Fig. 1. Treatment with TCA via oral intubation lasted for 28 days, with animal body weight and fasting blood glucose levels recorded during the experiment. The oral glucose tolerance test (OGTT) was executed on the 25th day according to the method of Irudayaraj et al. 26 . On the last day, the animals were placed on an overnight fast and thereafter placed under anaesthesia using xylazine/ ketamine (5:50 g/g).
Collection and preparation of samples. The experimental animals were sacrificed via cardiac puncture, collected blood was transferred into a heparin bottle and separated to obtain plasma and erythrocytes 27 . The liver was excised, prepared and stored as previously reported 25,28 . The excised and fat free pancreas was fixed in 10% formal saline for histology. www.nature.com/scientificreports/ Diabetic and biochemical parameter assays. The diabetic, liver and kidney function parameters were assessed in the plasma and liver. The plasma activity of α-amylase, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and plasma concentration of glucose (GLUC), bilirubin (BIL), albumin (ALB), urea, creatinine (CREA), calcium (Ca), sodium (Na), bicarbonate (HCO 3 ) and triglyceride (TRIG) were analysed using Randox and Teco diagnostic kits according to the manufacturers' instructions. The blood urea nitrogen (BUN) was calculated, taking into cognizance that 1 mg urea corresponds to 0.467 mg of urea nitrogen as stipulated in the manufacturers' instructions. Insulin (INS) concentration, α-amylase (AMY) and dipeptidyl peptidase IV (DPP-IV) activity in the plasma were assayed using Hangzhou Eastbiopharm ELISA kits, while protein (TP) was evaluated according to the method of Lowry et al. 29 .
Hepatic glycogen (GLYC) concentration was assessed following the method of Aba 30 with slight modification. Briefly, 200 mg of liver tissues were homogenized in 5 mL of ice-cold KOH (30%) and boiled at 100 °C for 30 min. The homogenates were precipitated with 95% ethanol in a ratio of 1:5, five times. The residue was then resolubilized in 500 µL of water. One hundred μL each of the resolubilized samples was pipetted into tubes containing 5 mL of freshly prepared Anthrone reagent. The contents were mixed thoroughly, covered and immediately boiled for 20 min. to induce colour development. Tubes were cooled and absorbance read at 625 nm using a spectrophotometer (Thermo Scientific GENESYS™ 10S UV-Vis Spectrophotometer, USA).

β-cell function and insulin resistance indicators. Evaluation of homeostasis model assessments for
β-cell function (HOMA-β) and insulin resistance (HOMA-IR) were determined by the methods described by Mohammed et al. 31 . Quantitative insulin sensitivity check index (QUICKI), composite insulin sensitivity index (CISI) and McAuley index (MI) were calculated according to the method described by Sasidharan et al. 32 , Sahin et al. 33 and Tsai et al. 34 respectively. These indicators were calculated using the following formulas shown in Eqs. 1-5:  Table 1 for first-strand cDNA synthesis, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene 21 . PCR products were run on ethidium bromide-stained agarose gel (1.5%) (Sigma Aldrich, Germany) in triplicates and viewed under UVP BioDoc-It Imaging system (Upland, CA, USA).
Pancreatic histology. The excised pancreatic tissues were histologically assessed as previously described 25,28 .
Graded concentration of ethanol was used to dehydrate the excised pancreas fixed in forrmalsaline. The pancreas were then cleaned and cleared using xylene, impregnated and embedded in parraffin wax, mounted on microscopic slides, sectioned to about 5 μm and stained using haematoxylin and eosin (H&E). Slides were observed using a slide scanner (Leica SCN 4000, Leica Biosystems, Germany) and interpreted.
Statistical analysis. Data are expressed as means ± S.E.M. Significant differences between multiple groups were determined using Duncan's multiple range test. p < 0.05 was considered as the significant value. All analyses were conducted using IBM SPSS v25 (IBM Inc., New York, USA) and Microsoft Excel 2019 (Microsoft Corporation, Redmond, Washington, USA) software packages.

Results
TCA treatment restores animal weight loss, plasma glucose level and clearance. Rats fed HFD significantly (p < 0.05) gained more weight than those fed standard rat chow for the first eight weeks. Induction of diabetes using STZ led to significant (p < 0.05) weight loss in HFD/STZ-induced diabetic rats but administration of TCA significantly (p < 0.05) reduced the weight loss in a dose-dependent manner that was significant (p < 0.05) than glibenclamide at the highest dose ( Fig. 2). During the intervention period, glibenclamide and TCA treatment groups showed a significant (p < 0.05) dose dependent decrease in blood glucose from the 12th day up until the end of the experimental regime compared with the untreated diabetic rats. The highest dose (800 mg/kg) of TCA decreased blood sugar at a rate which was comparable with glibenclamide ( Fig. 3). In Fig. 4, induction of diabetes significantly affected (p < 0.05) the glucose tolerance of the untreated diabetic group compared with the normal group. In contrast, glibenclamide and TCA administration significantly (p < 0.05) improved glucose tolerance compared with untreated rats. The glucose tolerance of diabetic rats treated with the highest dose (800 mg/kg) of TCA and glibenclamide were significantly comparable to each other at 60 and 120 min.  www.nature.com/scientificreports/ 10.0 and 14.8%, respectively) and hepatic TP (26.6, 21.0 and 25.4%, respectively) concentrations of the diabetic treated rats were significantly (p < 0.05) improved compared to the untreated diabetic rats (Fig. 5).
TCA treatment reverses disrupted mRNA expression. Induction of diabetes significantly (p < 0.05) reduced the expression of hepatic IRS-1, GLUT-4, P13K and AKT mRNA in the diabetic rats compared with normal rats. Treatment of diabetic rats with glibenclamide and TCA significantly (p < 0.05) improved the expression of these genes in the experimental group compared with the untreated diabetic animals. However, there was no significant (p > 0.05) change in DPP-IV mRNA expression even after the onset of diabetes and subsequent glibenclamide and TCA treatment ( Fig. 7 and S1).
TCA treatment repairs damaged pancreatic histology. Histopathology of pancreatic tissues revealed distinct exocrine acini with islets, blood vessel intralobular and interlobular duct in normal rats, glibenclamide  www.nature.com/scientificreports/ and TCA 800 mg/kg bw treated rats. For the untreated diabetic rats, vascular congestion significant with pancreatitis, acini and intralobular duct were aggregated with inflammatory cells and blood vessels congested with red blood cells were observed. In the pancreatic tissues of 400 mg/kg bw TCA-treated rats, a moderate aggregate of inflammatory cells were observed in the intralobular and interlobular duct (Fig. 8). www.nature.com/scientificreports/

Discussion
Rats that have diabetes induced by STZ exhibit austere body weight loss. This observation has been attributed to the rapid catabolism of structural proteins due to carbohydrates being unavailable to be utilised as a viable energy source 35 . This study further corroborates previous reports as treatment with STZ led to a loss of body weight in diabetic rats 36,37 . The reversal of this anomaly upon treatment with TCA suggests the protective property of the extract and its phytoconstituents on structural proteins, thereby preventing their degradation. OGTT is a more dependable assessment of detecting early glucose homeostasis abnormalities than fasting blood glucose as it depicts blood glucose uptake and hepatic gluconeogenesis after a glucose loaded/rich meal 38 . Excessive time-dependent elevation of blood glucose level indicated impaired glucose tolerance. The reduction observed after extract treatment indicated the mitigating effect of TCA on impaired glucose tolerance, further confirming the ability of the extract to improve insulin sensitivity in the tissues 38 . TCA also reduced the blood glucose levels monitored daily, suggesting glucose homeostasis was being restored to normal. This finding was similar to previous works where Xylopia aethiopica and Aframomum melegueta fruits were used 31,39 . The observed increase in plasma urea, BUN and decrease in plasma Na, Ca and HCO 3 after induction of diabetes is similar to the report of Ekperikpe et al. 40 . Similarly, the damage to the kidney, as observed in diabetics, is characterised by distortions in electrolyte homeostasis, giving rise to increased metabolites excretion above normal thresholds 41 . The induced loss of serum Na + and K + levels during diabetes onset is majorly due to dehydration 42 . Diabetic ketoacidosis is also a condition that arises due to the inability to utilize glucose for energy production hence generating ketone bodies and elevating acidosis increasing serum Clwhile reducing HCO 3 levels. The diabetic condition brings about a derangement in glucose production pathways leading to blood acidification, causing acid-base derangement with a subsequent increase in Cllevel to balance it 36 . Inhibition of the renin-angiotensin-aldosterone system as experienced in many endocrine systems may also play a role in electrolyte imbalance. Increase in plasma urea concentration is a known hallmark of diabetes nephropathy, as similarly observed in this study 43 . TCA administration modulated these observed electrolyte imbalances, suggesting it can restore tubular function in the kidney of diabetic rats and electrolyte homeostasis by attenuating diabetes-related hyponatremia, ketoacidosis and osmotic diuresis 44 . The quick attenuation of ketoacidosis would suggest a T2DM-related ketoacidosis as its resolution is significantly shorter than in T1DM 45 . The observed T2DM-associated ketoacidosis in our study was similar to previous studies where IR leads to increased HCO 3 levels and plasma α-amylase activity 46  www.nature.com/scientificreports/ The ability of the body to stimulate the uptake, utilise and store glucose through the action of insulin is a critical factor in maintaining a healthy blood glucose level 48,49 . The observed rise in blood and hepatic glucose with a concomitant rise in plasma insulin concentration, plasma α-amylase and DPP-IV activities in diabetic rats was consistent with the findings of other researchers who used the HFD and low dose STZ diabetes induction model 26,46,[50][51][52] . Interestingly, some other studies have reported insulin levels to be lower in the HFD/low dose STZ diabetes induction model despite the spike in blood glucose levels [53][54][55][56] . Diet manipulation to increase body fat content (HFD) combined with low dose administration of STZ induces insulin resistance and significantly www.nature.com/scientificreports/ elevates blood glucose levels 56 . The excessive intake of diet rich in fat also accelerates the release of insulin from the β-cell of the pancreas 46 . It is a model used to conveniently induce both hyperglycaemia and metabolic profiles in rats exhibiting close similarity to the clinical progression of T2DM in humans 57 . Protein and glycogen storage impairment have been reported to have a positive association with diabetes and an increase in hepatic glucose concentration. TCA's reversal of these abnormal conditions could be attributed to its ability to increase tissues insulin sensitivity thereby increasing the ability of the organs to utilise glucose for energy generation, in the process reducing plasma glucose concentration, insulin concentration, α-amylase and DPP-IV activity 58 . Reduced www.nature.com/scientificreports/ hepatic glycogen synthesis in the diabetic state has been attributed to irregular catabolism of hepatic glycogen stores and impairment of signals that stimulate hepatic glucose uptake and storage 30 . Plasma and organ protein levels are affected when there is insufficient glucose utilisation as the main source of energy generation 59 . The utilisation of glucose by the tissues will lead to increased generation of ATP, which may be responsible for the anabolic effect of insulin on protein metabolism retarding the observed reduction of plasma and organ protein concentration as well as the restoration of normal synthesis and storage of glycogen in the liver 60 .
The restoration of glucose homeostasis to normal may be attributed to the upregulation in IRS-1, GLUT-4, PI3K and AKT mRNA expression in the liver, which was reduced during the diabetes onset 61 . In normal physiology, insulin binding to the insulin receptor leads to its phosphorylation and initiation of insulin action. Activation of IRS-1, PI3K and AKT in a sequential manner leads to GLUT-4 vesicle translocation to the cell membrane, which finally induces glucose uptake in tissues as the concomitant effect of these chains of metabolic processes 12   www.nature.com/scientificreports/ vesicles' translocation, leading to the inability of tissues to take up glucose 62 . The induction of IRS-1/PI3K/Akt pathway upregulation concomitantly enhancing hepatic glucose uptake may be the attributable molecular mechanism of TCA in improving overall glucose homeostasis and overcoming insulin resistance in diabetes 63 . The molecular mechanism of action of TCA on HFD/low dose STZ diabetic rat model was first reported by our team. These biochemical observations were further corroborated with the results for indices of β-cell function and insulin resistance. Induction of diabetes reduced QUICKI, CISI, HOMA-β and McAuley indices in the rats with an increase in HOMA-IR as a consequence. TCA treatment attenuated these abnormal values, suggesting TCA's antidiabetic action could be attributed to the reversal of insulin resistance, partial pancreatic beta-cell dysfunction, and decreased insulin sensitivity. Mohammed et al. 39 and Mudi et al. 64 had the same findings on HFD/ STZ induced T2DM rats model using Xylopia aethiopica fruit acetone fraction and Aegle marmelos fruit and leaf aqueous extract, respectively, thus corroborating the study. These indices are used to precisely determine insulin sensitivity, resistance, β-cell function and glucose-insulin homeostasis, both clinically and epidemiologically 65 . The reversal of damaged pancreatic histology by TCA might suggest its ability to regenerate distorted pancreatic tissues.
In summary, this study shows that TCA ameliorates diabetes and its resultant downstream effect in high fat/ low dose streptozotocin-induced diabetic rats to improve dysregulated glucose metabolism. The mechanisms at which these ameliorating effects are achieved may be via GLUT-4, IRS-1, PI3K and AKT gene upregulation which may probably enhance GLUT-4 translocation, reduce insulin resistance and induce PI3K/AKT signalling pathway (Fig. 9). Hence, TCA could be exploited for efficient antidiabetic agents to remedy diabetes and restore glucose metabolism. Inability to study the protein expression of evaluated genes to corroborate the gene expression result and other mechanisms through which TCA might have exerted its antidiabetic effect were some limitations of this study. In addition, random sampling of the hepatic tissue might have been a confounder in glycogen assessment. Therefore, novel mechanisms of TCA and further molecular assessment of protein expression should be studied to understand its antidiabetic action fully.  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.